Isolation of <i>Synechocystis</i> Mutants Overproducing Mannitol Directly from CO₂ via Laboratory Evolution under Increasing Salt Concentration

Mannitol is a naturally occurring C(6) polyol with a wide range of applications in the food and pharmaceutical industry. In a previous study, mannitol production was achieved via the direct conversion of CO₂ in <i>Synechocystis</i> sp. PCC6803. However, a major barrier to future applications of this strain was its low production rate. In this study, three mutants were isolated after 164 generations of adaptive laboratory evolution under salt stress. These mannitol overproducing mutants were able to produce 27.71 mg L^-1 OD₇₃₀^-1 mannitol under 350 mM salt stress when the OD₇₃₀ reached 2, roughly 24 times higher than their parental strains. Whole-genome resequencing was then performed, revealing mutations in 2 genes─<i>pnp</i> and <i>sigA</i>/<i>rpoD1</i>─of the overproducing mutants when compared to the parental strain. Of these genes, <i>pnp</i> which encodes for polyribonucleotide nucleotidyltransferase was found to negatively affect mannitol production in cells via reverse engineering methods, in which the (partial) removal of <i>pnp</i> alone resulted in a 6.4-fold increase in the mannitol production. The work reported here substantially advances mannitol production capabilities in engineered <i>Synechocystis</i> sp. PCC6803 strains through adaptive evolution but also highlights the previously unrecognized negative regulatory role of <i>pnp</i> in mannitol synthesis.